Borrelia spirochetes causing relapsing fever (RF) can persist in an infected host by antigenic variation of Vsps and Vlps, two families of variable, immunodominant and thus serotype-defining surface lipoproteins. Vsps and Vlps induce a T-independent neutralizing IgM response, which leads to complement-independent elimination of that specific serotype. Yet, these antibodies do not protect from subsequent spirochetemia of other serotypes. Highlighting an additional evasion strategy, one of two Vsps with different binding affinities to glycosaminoglycans (GAGs) has been implicated in targeting RF Borrelia to the central nervous system, an immunopriviledged site. The overall objective of this proposal is to gain an in-depth understanding of these pathogenic processes by defining the structural and functional basis of Vsp- and VIp-mediated immune evasion and tissue tropism. Their demonstrated overlapping role in both mechanisms lead us to initially focus on the Vsps. Our preliminary data indicate that Vsps share a conserved dimeric and alpha-helical structural fold, with hypervariable loops connecting the alpha helices. We hypothesize that these variable and membrane-distal loops (i) form the Vsp-specific antibody epitopes, and (ii) also confer the surface properties involved in tissue localization. To significantly facilitate our studies, we have developed a novel surface display system, which allows us to stably express and present functional Vsps and Vlps on the surface of the Lyme disease (LD) spirochete Borrelia burgdorferi. We will now apply this system to test our hypothesis and have formulated two major specific aims: 1. to define the neutralizing antibody epitopes of Vsp proteins: Based on the three-dimensional structure of two Vsps and using specific IgG and newly generated IgM monoclonal antibodies, we will map antibody epitopes by analyzing Vsp escape mutants and antibody interactions with Vsp chimeras and point mutants. 2. to define the Vsp domains interacting with GAGs: Chimeras and point mutants of two Vsps will be examined for binding to purified GAGs, glial and endothelial cells as well as for neurotropic characteristics. These studies will significantly increase our understanding of the pathogenesis of Borrelia infections, and shed more light on the mechanisms of antigenic variation and immune evasion of microbes in general. Furthermore, they will yield important clues for the design of future intervention strategies.